The goal of the research proposed here is to develop improved adenoviral vectors that can be used for gene transfer to the liver for the treatment of hepatic deficiencies. Although adenoviral-mediated gene transfer to hepatocytes in vivo is very efficient in animal models, improvements are needed because of the transient nature of gene expression and the inability to perform repeat transduction. The specific aims are to: 1) Develop a recombinant adenoviral vector deficient in E4 gene function. The E4 deficient vectors besides allowing more room for cloning exogenous genes, should lead to less endogenous adenoviral gene expression in transduced cells. As a result, there may be less interference with host cell gene regulation and less probability that the vector will produce antigens that ultimately will limit the life-span of transduced cells in vivo. 2) Establish the role of the antigen-specific immune response in a) the loss of adenoviral-mediated gene expression in hepatocytes and b) the inability to achieve secondary transduction of hepatocytes after adenoviral-mediated gene transfer in vivo. Delineation of these immune responses will allow for rational design of vectors which are non-immunogenic. 3) Investigate the significance of individual proteins encoded in the adenoviral E3 region in recombinant adenoviral vectors after in vivo hepatocyte gene transduction. Constitutive high level gene expression of some of the E3 gene products may protect transduced cells from immune-mediated destruction. We propose to directly address these issues in vivo. 4) Investigate adenoviral-mediated gene transfer into biliary epithelial cells to develop this as a method for the treatment of a number of medical disorders. 5) Evaluate the biliary tract as a means of delivering genes into hepatocytes in vivo. This mode of gene delivery has potential advantages over the vascular routes of delivery and will be explored as an alternative to current methods. The successful completion of this project will have general applications to all cell types that are currently being targeted by adenovirus as a vehicle for gene transfer. Using the liver as a target organ will have general application for gene therapy for a large number of genetic disorders resulting from hepatic deficiencies.